A recent focus of this project has been the role of the 3' long open reading frame of the human T-cell leukemia virus type-I (HTLV-I) which encodes a 40-Kd protein (p40x). This protein positively regulates transcription directed by the HTLV-I long terminal repeat (LTR) in a phenomenon known as trans-activation. We have succeeded in expressing the complete p40x coding sequence in E. coli and in a baculovirus vector. Both p40x proteins are capable of stimulating transcription from the HTLV-I LTR. Significant purification of the p40x proteins has been achieved. We have been unable to attribute any sequence-specific DNA binding properties to p40x, suggesting that the protein activates the HTLV-1 promoter in an indirect fashion using cellular transcription factors. Our objective is to understand the biochemical mechanism of trans-activation by the p40x protein and the involvement of cellular transcription factors in this process. Distinct transcriptional regulatory sequences located in the upstream sequences of the HTLV-I LTR have been identified and chemically synthesized. These sequences, which have the properties of enhancer sequences, have been cloned and are trans- activated by the HTLV-I p40x protein. Using a novel DNA-protein cross-linking protocol developed in this laboratory, we have identified cellular factors that interact with the 21 bp p40x- responsive sequence. We have demonstrated, by mutational analysis, that the binding of the cellular proteins in vitro correlates with the in vivo biological activity in response to p40x trans-activation.